A Role for Iron in an Ancient Carbonic Anhydrase

Brian C Tripp,Caleb B Bell,Francisco Cruz,Carsten Krebs,James G Ferry

doi:10.1074/jbc.m311648200

Brian C Tripp, Caleb B Bell + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m311648200

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Abstract

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe2+, which yielded Cam with an effective kcat that exceeded that for the Zn2+-reconstituted enzyme. Mössbauer spectroscopy showed that the Fe2+-reconstituted enzyme contained high spin Fe2+ that, when oxidized to Fe3+, inactivated the enzyme. Reconstitution with Fe3+ was unsuccessful. Reconstitution with Cu2+, Mn2+, Ni2+, or Cd2+ yielded enzymes with effective kcat values that were 10% or less than the value for the Zn2+-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2+-reconstituted Cam. The results identify a previously unrecognized biological function for iron.

Highlights

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (␣ class) and plants (␤ class); two additional classes (␥ and ␦) were discovered recently
Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2؉-reconstituted Cam
The crystal structures of Zn2ϩ- and Co2ϩreconstituted Cam show the metals coordinated to the nitrogen atoms of histidines in all three active sites [18]

Summary

A Role for Iron in an Ancient Carbonic Anhydrase*

From the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. CAs are hyperproduced in E. coli cultured in medium supplemented with Zn2ϩ and purified aerobically with buffers containing Zn2ϩ These procedures have the potential to bias the incorporation of Zn2ϩ in vivo during synthesis of the enzyme or by in vitro exchange of another metal for Zn2ϩ during purification. We show that replacement of zinc in Cam with ferrous iron yields an enzyme with effective kcat values exceeding zinc-containing Cam, identifying a previously unrecognized biological function for iron

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RESULTS

DISCUSSION